CCR5 Ligand Levels and Immune Response to HIV Vaccines [Revised Proposal]

ZRG1 VACC (01) 3 1 RO1 A1052038-01A1
GARZINO-DEMO, A
trials of an HIV vaccine that might show clinical efficacy have started and when they do results will not
be available for over 5 years.
APPROACH: The revised application will address the question of developing novel assays for
evaluation of HIV vaccine efficacy. The overall approach is to develop ELISA formats for measuring
the proportion of full length RANTES and LD78B and correlate levels of HIV antigen inducible
chemokine levels produced by PBMC from vaccine recipients and other measures of cellular and
humoral immunity. There are three specific aims proposed in this application.
In aim one, the investigators will develop ELISA based assays for LD78B and -2 RANTES which are
the potent alpha viral chemokine isoforms. The ability to define the contribution of each of these
isoforms in HIV pathogenesis is clearly important. The issue is will an ELISA format provide sufficient
sensitivity, if low levels of the chemokine are produced following antigen stimulation. The other issue is
that this approach will not provide insights into which cells are capable of producing the individual
chemokines. The investigators have dealt with the issues of producing antibodies for the ELISA but
this assay format is really not an advance over existing assay formats. The investigators also propose
to develop RNA based assays to quantify LD78B and -2 RANTES. The issue here is that RNA levels
being equivalent to protein is not always the case. The investigators need to provide preliminary data
to show this is true and that RNA detection might be a relevant marker for monitoring vaccine response.
No preliminary data is present in their tetanus immunization system comparing RNA and protein levels.
They also discuss using NASBA based assays and simply provide literature references for the
methodology. Further preliminary studies need to be performed to validate the value of this approach.
In specific aim 2, the application proposes to characterize and standardize a panel of ELISAs for CCR5
ligands that can be used to analyze primary blood samples that secrete cytokines. This aim will utilize
PBMC samples obtained from seronegative volunteers at 6-month intervals and stimulated in vitro with
tetanus toxin, Candida albicans and PHA. Inducible levels of chemokines will be compared to 3Hthymidine uptake. Fresh and frozen samples will be compared. This approach should yield
interpretable data, but its comparability to HIV vaccine recipients is not very strong. A more effective
approach would be to evaluate subjects receiving vaccines such as hepatitis B that may give results
more compatible to HIV vaccines. Indeed one could obtain samples from seronegative vaccines.
Thousands of seronegative individuals have received experimental HIV vaccines and those cells could
be obtained and evaluated. Indeed significant amounts of cellular and humoral immune studies will
have been performed on such samples for comparison. The other concern is the investigators
assumption that freezing will not impact chemokine activity. It is very clear that freezing diminishes
APC function and subsequently will effect antigen driven proliferations. There is also some question
about the statements on methods of cell freezing. Many large multicenter studies (MACS, WITS,
WIHS, ACTG) have shown no difference in programmable freezing versus freezing boxes. The
investigators make many assumptions that have not been borne out by established studies.
For specific aim 3, the investigators will study antigen specific induction of chemokines in samples
obtained from subjects enrolled in vaccine trials. The focus of the application is to utilize samples from
phase I and I1 clinical trials of vaccines based on canary pox and other vectors. This is a limited
approach as currently the most immunogenic vaccine candidate is a DNA vaccine. Indeed phase Ill
trials in the HIV Vaccine Trials Network were stopped because recombinant canary pox HIV candidate
vaccines did not achieve sufficient immunogenicity in phase II trials. The real issue is that the authors
will simply end up correlating an antigen induced chemokine response with other cellular and humoral
assays, which will not validate chemokines as a correlate of protective immunity.
INNOVATION: There is very little innovation in this application. The assays being developed are
standard ELISAs and the resulting data will simply correlate chemokine levels with cellular and humoral
immune markers. The study will not validate chemokines as correlates of protection.